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Team:Cornell/project/wetlab/lead
From 2014.igem.org
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<h1 style="margin-top: 0px;">Construct Design</h1> | <h1 style="margin-top: 0px;">Construct Design</h1> | ||
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In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene CPB4 codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of <i>Bacillus spp.</i> found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant <i>Nicotiana tabacum</i>. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications [1]. | In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene CPB4 codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of <i>Bacillus spp.</i> found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant <i>Nicotiana tabacum</i>. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications [1]. | ||
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| + | Cells successfully expressing CBP4 should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460005 in the amp<sup>r</sup> plasmid pUC57 were grown for a 24 hour period in LB with 1 mM, .1 mM and .01 mM Pb. | ||
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| + | Part BBa_K1460005 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-YMT) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted by 1/2 with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. | ||
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<h1 style="margin-bottom: 0px">References</h1> | <h1 style="margin-bottom: 0px">References</h1> | ||
Revision as of 19:38, 11 October 2014
Wet Lab
Construct Design
In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene CPB4 codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of Bacillus spp. found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant Nicotiana tabacum. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications [1]. Our first lead transporter construct (BBa_K1460005) consists of the constitutive Anderson promoter and the CPB4 gene for constitutive expression of the heavy metal membrane transporter and uptake of lead. The primary construct for lead sequestration (BBa_K1460008) consists of this first lead transporter construct put upstream of our metallothionein construct with the T7 promoter and GST-YMT. This construct allows for the constitutive expression of the lead transporter as well as the inducible expression of the metallothionein in BL21 by arabinose activating the araBAD promoter and allowing expression of the highly active T7 polymerase. This allows for our bacterial strains to grow to stationary phase before being induced to produce metallothioneins and being used to sequester lead.
BBa_K1460008
Results
Cells successfully expressing CBP4 should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, E.coli BL21 and engineered BL21 with part BBa_K1460005 in the ampr plasmid pUC57 were grown for a 24 hour period in LB with 1 mM, .1 mM and .01 mM Pb.
Part BBa_K1460005 in pUC57 was co-transformed with part BBa_K1460001 (GST-YMT) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted by 1/2 with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab.
References
- Ref 1
- Ref 2
- Ref 3
